This invention relates to modular devices and methods for concentrating and/or purifying macromolecules in a solution as well as apparatuses including such devices.
The basic device comprises essentially a pressure resistant concentration chamber which can be provided with modular accessories adapting the device for use with a choice of gas pressure, vacuum, centrifugal forces or a combination of gas pressure and centrifugal forces.
The pressure resistant concentration device can be used either in a centrifuge, on a laboratory agitator or as a free standing pressurised or vacuum driven filtration cell. Interchangeable accessory-modules makes it possible to process the solution by means of e.g. two stage filtration, a combined diafiltration and concentration procedure or by means of one or more solid phase extraction steps which can be carried out in combination with sample concentration.
Many biotechnology laboratories use micro- and ultrafiltration methods in the processing of biological solutions. As examples, filtration is used as a sterilising step to remove bacteria, as a clarification step to remove suspended solids and contaminants, as a concentration step for proteins and other macromolecules or as a purification step to eliminate unwanted micromolecules such as salts. Alternative membranes and porosities are used to suit specific applications and process requirements.
Filter elements are mounted in a broad array of holders and forces due to liquid or gas pressure, vacuum or centrifugal forces are used to transport the filtrate through the filter element. This results in a very broad offer of products to the research scientist who must invest in many alternative products and processing techniques to cover a broad range of filtration requirements.
Amongst the more popular techniques for filtration of laboratory biological samples are centrifugal-force-techniques and a technique making use of gas pressurised cells. For single samples, stirred cells are more popular whilst multiple samples are more frequently processed in centrifuges where more than one sample is usually required to balance the centrifuge. As up until now, the filter holders have been incompatible for use in both a stirred cell and centrifuge, laboratories have had to purchase both types of filter holders to fully cover their requirements.
Pressurised cells frequently use a stirring bar mechanism to maintain the solution being processed in suspension and in order to inhibit the build-up of macromolecules on the membrane surface which would result in reduced filtration speed. Whilst stirring mechanisms are quite effective for this purpose, they also induce shear forces which can denature macromolecules and reduce the biological activity of the solution.
In order to clear a higher proportion of contaminating micromolecules during the filtration process, an additional container could be linked by a tube to the filtration cell feeding a buffer solution to the filtration cell at the same speed as effective filtration. The filtrate is carried by another tube to another adjacent container. Several tubing connections and a large amount of bench space is required to accommodate the various containers.
In centrifugal filtration procedures, during the first stages of filtration, pressure across the membrane is high due to the high centrifugal force on the full initial volume of the sample and filtration speed is therefore at its peak. However, as filtration progresses and the solute above the membrane is reduced, filtration speed is also reduced and achieving a high level of filtration becomes very time consuming. Whilst a proportional increase in centrifugal speed can theoretically maintain a constant trans-membrane pressure that is usually not possible due to limitations in the maximum speed of centrifuges and the mechanical limitations of the filter holder.
Another problem with centrifugal ultrafiltration is that the concentration device cannot be sealed from the outside environment as an air passage is necessary at the head of the device to stop generating retentive vacuum as filtration progresses. This can cause the build up of aerosols during the centrifugation process and contamination of the centrifuge itself. This is a particular problem with toxic and dangerous substances. Whilst protective containers for the whole filtration assembly are sometimes used, these are expensive and reduce the capacity of the centrifuge rotor.
In order to reduce the number of filtration steps required in sample processing, some device holders can be stacked together to provide a double filtration during the centrifugal process. For example, a solution may pass through a first relatively coarse filter for clarification followed by the concentration of the molecules of interest by retaining them in an impermeable area on the edge of the second filter. The objective is to clear a maximum of the solute of interest through a relatively coarse first filter which is ideally positioned at 90xc2x0 to the vector force and then avoid the build up of the solute on the second tighter filter by positioning the second membrane element at an angle to the vector force. Up until now this technique has been limited by the fact that the surfaces of stacked filters have been arranged in parallel to each other.
In addition to filtration, chromatography techniques e.g. making use of solid phase extraction columns are sometimes used to complement filtration and achieve a higher level of sample purification. This is normally a separate step to filtration in the processing of biological samples and is therefore time consuming. Also, solid phase extraction columns are difficult to control as solution flow through the gel bed must be sufficiently slow to allow target molecules to be fully captured by the sorbent bed. In most cases this precludes the use of gas pressure or centrifugation because of excessive flow rates through the sorbent material. More common techniques such as the use of a manually controlled syringe is time consuming and often inconsistent; or alternative the use of vacuum filtration manifolds require additional capital investment and longer set up times.
One object of this invention is to provide a filtration cell or device which can be used in either a centrifuge or as a pressure driven free standing unit thereby eliminating the need for specific single and multiple sample filtration devices.
It is another object of this invention to provide a pressure driven filtration cell or device which eliminates the need for a stirring mechanism for the purpose of creating turbulence, sample agitation being provided by an external shaking or vortex inducing action.
It is another object of this invention to provide a pressure driven filtration cell or device which requires reduced bench space and eliminates the need for tube connections to ancillary containers when the device is used in e.g. a diafiltration procedure.
It is another object of this invention to provide a pressure driven filtration cell or device which can be used in either a centrifuge or as a pressure driven free standing unit, which when used under gas pressure in a centrifuge, combines the filtrate driving forces from gas pressure and gravity for improved flexibility and control of the filtration procedure.
It is another object of this invention to provide a pressure driven filtration cell or device which can be used in either a centrifuge or as a pressure driven free standing unit, which when used under gas pressure e.g. in a centrifuge eliminates the need for an air passage between the sampler reservoir and the external environment.
It is another object of this invention to provide a pressure driven filtration cell or device which can be used in either a centrifuge or as a pressure driven free standing unit which comprises two membrane elements and in which the proportion of solvent to be retained by each filter element can be controlled.
It is another object of this invention to provide a pressure driven filtration cell or device which can be used in either a centrifuge or as a pressure driven free standing unit, which in addition to a filtration step can provide a solid phase extraction step whereby the speed of flow across the sorbent in the extraction step is controlled by the back pressure generated by the membrane element.
It is another object of this invention to provide a pressure driven filtration cell or device which can be used in either a centrifuge or as a pressure driven free standing unit, which has an integrated reservoir which feeds a washing solution at the same rate as contaminants are cleared through the filtration membrane. It is further an object of this invention to provide a modular device which alternatively can be set up and used in pressurised and non-pressurised mode.
It is also an object of this invention to provide new apparatuses including devices according to the invention in pressurised or non-pressurised mode.
It is further an object of this invention to provide new methods for processing liquid samples making use of devices or apparatuses according to the invention.